In the last twenty years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized. Our laboratory, in collaboration with others, undertook two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 100 regulatory RNAs that are now identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively, although some of these sRNAs also have additional roles. Our lab has studied a number of these sRNAs in detail. Each sRNA is regulated by different stress conditions, suggesting that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act. The lab continues to investigate the in vivo roles of small RNAs, identifying the regulatory networks they participate in and their roles in those networks. Many targets of sRNAs are regulated by more than one sRNA, and our earlier approaches have not allowed us to identify all of the sRNAs regulating a given target. In addition, regulatory proteins expressed at low levels or under special conditions may not be identified as targets for a given sRNA if their transcripts are not reasonably abundant. We have developed and made use of an approach to deal with these limitations. Translational fusions can easily be created to possible target genes, identified based on the ability of their mRNA to bind Hfq, among other criteria, and the fusions can be rapidly screened with a library of plasmids, each expressing a different sRNA. Bacterial behavior can similarly be screened. We have applied this approach to the study of bacterial motility and biofilm formation, and have also examined the sRNAs regulating a set of important transcriptional regulators and proteins involved in stress responses. Our studies of sRNAs that, when overproduced, modify biofilm formation allowed us to identify alternative pathways leading to formation of biofilm. mutS, encoding a component of the mismatch repair system, was found to be regulated by a small RNA, ArcZ, and, somewhat surprisingly, directly by Hfq in the absence of sRNAs, dependent upon sites in the mutS 5'UTR. Mutation of these sites leads to increased levels of MutS protein in stationary phase cells and decreased mutagenesis, demonstrating the role of post-transcriptional regulation in allowing mutagenesis as cells run out of nutrients. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins has been found to regulate levels of the signaling molecule acetyl phosphate. Lessons learned from this project suggest the importance of many other previously unappreciated sRNAs made from 3' UTRs. The action of these small RNAs depends on the RNA chaperone Hfq, a protein with homology to the Lsm and Sm families of eukaryotic proteins involved in RNA splicing and other functions. Hfq binds both to sRNAs and to mRNAs, and stimulates pairing, but exactly how it does this has been clear. In a series of studies, in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out an in vivo dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. We have found that the Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. All of these sRNAs depend on the known sRNA binding site on the proximal face of Hfq for in vivo stability. Class I sRNAs are rapidly degraded when used, most likely dependent upon pairing; their targets bind to the distal face. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites in Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on differentsRNA:mRNA pairs. The C-terminus of E. coli Hfq (CTD) is unstructured, and its role has been unclear. In collaboration with S. Woodson, we have defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In a collaboration with E. Nudler, a novel role of sRNAs in regulating transcription elongation by controlling the access of a transcription termination factor Rho to mRNAs was described. This process is being further analyzed in the lab via a novel bi-functional fluorescent reporter that should allow us to distinguish effects of sRNAs on mRNA stability and effects on mRNA translation. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. Our focus is increasingly on the role of the sRNAs in complex bacterial behavior, investigations into the mechanism of sRNA function, and dissecting of novel mechanisms for regulating translation initiation. We have also returned to our interest in the regulatory cascade affecting capsule synthesis, in a collaboration with S. Buchanan and NCATs. The proteins in this cascade also regulate aspects of the bacterial response to membrane stress, are needed for in vivo establishment of commensal growth, and are important virulence factors in Klebsiella. Studies on the Interactions of the components of the regulatory cascade have changed our understanding of signal transduction through this system. We have developed an efficient assay for screening for small molecules that activate or inactivate the cascade, and are analyzing the initial molecules identified by NCATS through this screen. The long-term goal of this is to investigate the development of novel antibiotics that act by perturbing this important regulon.